-went down to Koshland to do confocal with Scott from the king lab. Could only load 30 ul of sample at time. Looked at NLS and 51 after induction fifteen minutes after feeding.
-saw one possible hit, but set up wasn't optimal
-choanos were suspended in low melt temp agarose.
-probably better to use scope in Stanley.
-lysis looks like it occured in pmll KC. miniprepped the plasmid, but probably will not use because it took so long. Also streaked out control, and will retest for lysis
-for the future: JTK030 and 159 have different copy numbers. Probably why timing is different for 159 with 2801 and NLS which is 030.
-New lifeact part 171 in 1600 still did not grow up

-started fresh choano culture by adding Kan to choano media culture.
-picked colonies of AKS NLS with 51 and 52 as well as 2801 with 51 and 52 to look at under Koshland confocal.
-checked on colonies of iGEM171 in 1600 (p15 spec). Did not grow up. Talk to Tim.
-tested lysis of PCR of 51 digested and ligated in pmll KC. Did not appear to have lysed after 6 hours. kept in shaker

-did choano assays with 1934 NLS in JTK030 at induction time points +30, +15, 0, -15.
-Had dones more hits in 51 for 0 and -15 time points. could barely move in field of view without seeing a hit!
-52 did not have any hits at all.

Ran choano assay on just 1968 in '11' with plain '11' as control. Didn't really see any of the same successful choano phenotype after 3 hours after feed at 90,60 and 30 min induction points. In addition, adding more bacteria (5ul vs 1ul) doesn't really appear to help efficiency. Just see a lot of stuff lysing outside of choanos. May not be limiting factor.

Christoph ran lysis assays over weekend in LB/ASW and choano media. Said he saw lysis in all of them. Will run Tecan tomorrow to confirm.

Did microscopy assay of 51 and 52 induced 90, 75, 60,45,30,15 mins ahead of time after 24 hours. Did not see green phenotype anywhere, or really all that much green. Not a success, but not surprising considering that we don't really see lysis in ASW.

New Payload Work

Also created new assembly trees and Eco Bammed everything into pmll vectors.

Retransformed 41, 42, 45-52 and plated on AC for 41 and 42 and AK for 45-52 to avoid contamination issues. Asked Daniella to pick them for me tomorrow. Daniela said she would pick colonies for EcoBam stuff and do 2ab assembly on Friday. I'll do lysis testing.

Lysis

New Payload Work

retried Christoph and Conor's shortcut for assembly of the two new payloads by PCRing of PCON from Josh (plate 5, well c10) with phusion. Expected band length was 1000 bp. Cut and gel purified, and digested with Eco Bam. Also digested Lifeact GFP and RFP from Jin with Eco Bgl. Ligated Pcon eco bam into Eco/bgled lifeact rfp and gfp and transformed into Pir Lefty.

Lysis Testing

Amy ran lysis test and saw no lysis. Will rerun test for her in just TB tomorrow.

Choano Assay

Set up lysis assay for 47 and 48 with payload in LB vs in ASW, with Mc1061 with and without ATC as a control. Results were that both control and positive ATC OD dropped off at the same rate, possibly because the temperature control on the TECAN is at 26 degrees rather than 37.

Lifeact RFP didn't grow up very well so was unable to get it to grow up well enough to make comp cells. Will retransform next week.

Choano Assay

growth of cells in LB was poor, which confirms that life-time on each transformation is less than a week. Ideally within the first four days of life of the plate. Set up chonao assay again but saw no lysis. Will redo in TECAN tomorrow using TB to get better growth.

made competent cells for Lifeact GFP in JTK030 and Bjh1934 in JTK030.

Choanos

none of choano cultures, including 1:2 was at assaying density yet. Fed each 40 ul and let sit.

Choano Assay

Set up chonao assay. Washed saturated LB culture of 47 and 48 with payload delivery in choano media, ASW, TB and LB. Using 2 3 mL aliquots, added ATC to one tube and none to other to serve as control. DId not see lysis after 3 hours. Will grow up in LB and redo test on Thursday.

re-cotransformed JTK030 with Bjh1934 and pBca1256 for assaying this week. Transformed JTK030 with lifeact GFP and RFP and 1934to make comp cells.

IBB

Eco Bammed RFP Boo15 out of hybrid plasmid into KC, and transformed into MC1061 pir righty.

Quintara resequenced Rbs ibb, gfp and it looks perfect. Tim deleted the first three bp off the oligo, so that's why there is that deletion in the front of the rbs.

Choanos=

Choano Assay

ran assay of taking pictures of choanos eating just payload bacteria every five minutes for an hour. Saw photobleaching at a round minute 82. Arrived at scope 15 minutes after having fed choanos and were not seeing any rod phenotype. Hard to conclude the details of choano digestion because choanos keep moving slightly in and out of plain, messing up focus of FPs. Need to figure out how to fix choanos in space.

Lysis Assay

Did a loose assay to check if construct 47 and 48 without payload were lysing in ASW. Grew up in LB to saturation, washed and added ATC. Did not lyse. Picked and grew up colonies in TB to redo assay tomorrow. Will use just TB culture as a control, because it should lyse obviously within two hours according to Amy.

looked at chonaos at around the two hour mark. Very little fluorescence at all, only getting fluoresence when we bumped up the exposure to like 500 ms. Possible that the cells are lysing way before they are eaten. Chris says to try intervals between 15 minutes before to 15 minutes after feeding.

in assay, chase with less white ecoli.

from assay, can conclude that choanos complete digestion definitely within 2 hours, probably within 1.

no such thing as narchoanos. :( just choanos with two flagella.

IBB Work

miniprepped 3 of 4 colonies that weren't cotransformed for assembly without ATP and with DNTPs.

IBB Work

miniprepped RFP KA and mapped it to make sure it wasn't methylated at bgl and bam.

eco bam of rfp bam xho bgl xho. looks good

got BCA1092 from Jin and transformed it into righty.

miniprepped TIm's GFP.

started assembly of AC Tim's GFP with CK rbs ibb.

sent RBS ibb in for sequencing.

Lifeact

Lifeact RFP transformations came up green, which means i used the wrong DNA. Redid Eco Bam and transformed into MG1655.

Choano Assay

had Conor plate my transformations, but I'm worried about them actually being co transformed because top plated spec and you can see that they are all growing up around the edges, where there might not be spec. Fishy. Redid cotransformations with 1 ul and 3 ul of straight DNA of each.

observed after 1 hour, 4 hours. Still too much green bacteria outside cells, looks like they are still eating. Lots of rod-shaped. Strangely, not a lot of degradation of gfp after 1hour in cells without nh4.

lifeact: after 1 hour, cells are green, can see rod shaped bacteria. After four hours, choanos look pretty empty (digested?) Interesting to note that several times we observed choanos with extremely round dot of green adjacent to them? Also saw balls of green in their bodies. Actin is balling up?

IBB

did Eco/Bam of Lifeact RFP into 1256 (f4 on TIm's stock plate 1) background will be white. Will transform tomorrow.

PDD Subgroup Work

started Eco Bam transfer of 1882 part for lifeact tree into AK.

sent Ptet+Lifeact in for sequencing. We suspect it is bad because one of the inputs was in KA, which was actually AK.

Talked over results of minimal media testing with Jin. Results were ambiguous because there was inconstancies between the duplicates. Additionally, Jin thinks that Mg in the choano media may be obscuring the results. One thing he observed that he wants to investigate further is that he thinks 37 C might be slowing down the choano's digestion.

Split chonaos 1:2 so they will be ready for assaying on Thursday

Started transformations of 3s for practice assay on Thursday Morning. Talked to Jin. He said transformations are good for about 3 days on plate.

PDD Subgroup Work

direct Jin to confirm which of the combinations is actually working.

remind Tim to email Jin about scope.

Life Act

Even after 24 hours, the bacteria from the colony PCR still did not grow up well. Only saw growth for a couple. Miniprepped colonies 6, 9, 19, 20. Restriction mapped Bam, Eco, excpected band length of 167 and 2700

controls for JTK030 looked good with no growth on Kan, Amp, Cam, good growth on LB, and a little growth on Spec, which is the normal result because of mutations in the bacteria. Those are labeled with double black stripes, in a box in the back of the -80 in the 2nd row from the top.

Kan LB Cam Amp Spec

picked and grew up the results of PCA for the new nuclear localization tag. They had plenty of colonies, with only one background green colony. Will miniprep them tomorrow.